Creative Proteomics offers an integrated solution for the identification of low abundance proteins in complex biological sample through 2D electrophoresis.
In two-dimensional gel electrophoresis, proteins are first separated by their pI in isoelectric focusing and then further separated by molecular weight through SDS-PAGE, thus the sample proteins are distributed across the two-dimensional gel profile. This technique expands the number of proteins that could be identified, provides more efficient data and detailed information for proteomics analysis.
Two-dimensional gel electrophoresis, abbreviated as 2-DE, is a form of gel electrophoresis commonly used to analyze mixture of proteins by two properties in two dimensions respectively.
2-D electrophoresis begins with 1-D electrophoresis and then separates the molecules by a second property in a direction 90 degrees from the first. In 1-D electrophoresis, the proteins separated in one dimension will lie along a lane, and then the molecules are spread out across in the 2-D gel. Generally, it is unlikely that two molecules will be similar in both two distinct properties, so molecules are more effectively separated in 2-D electrophoresis than in 1-D electrophoresis.
Isoelectric focusing (IEF) and Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis (SDS-PAGE) are preferred in 2-DE separation. In IEF/SDS-PAGE, the proteins applied in the first dimension will move along the gel andaccumulate at their isoelectric point; that is, the point at which the protein has a neutral charge.
Before separating the proteins by mass in the second dimension, they are treated with sodium dodecyl sulfate (SDS) along with reducing reagents which unfolds them into long, straight molecules bound with a number of SDS molecules. Since the SDS molecules are negatively charged, the result of this is that all of the proteins will have approximately the same mass-to-charge ratio as each other. In the second dimension, an electric potential is applied at a 90 degree angle from the first field. The gel therefore acts like a molecular sieve when the current is applied, separating the proteins on the basis of their molecular weight with larger proteins being retained higher in the gel and smaller proteins being able to pass through the sieve and reach lower regions of the gel.
After 2-DE analysis, a gel with proteins spread out based on their pI and molecular mass is obtained. These proteins can then be detected by a variety of means, but the most commonly used stains are silver and coomassie brilliant blue staining. In the former case, a silver colloid is applied to the gel. The silver binds to cysteine groups within the protein. The silver is darkened by exposure to ultra-violet light. The amount of silver can be related to the darkness, and therefore the amount of protein at a given location on the gel. This measurement can only give approximate amounts, but is adequate for most purposes. Other staining method such as coomassie brilliant blue combined with in-gel digestion is suitable for MS detection afterwards.
2-DE is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. This technique sorts proteins according to two independent properties in two discrete steps. Each spot on the resulting two-dimensional array corresponds to a single protein species in the sample. Thus thousands of different proteins can be separated, and information including the protein pI, the apparent molecular weight, and the amount of each protein is obtained.
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